Innovative Antigen Display with Self-Assembling Ferritin Nanoparticles

Introduction

In the rapidly evolving fields of immunotherapy and vaccine development, ensuring the effective delivery and display of antigens is crucial for stimulating robust immune responses. Traditional methods of antigen delivery often face challenges in ensuring proper assembly, stability, and targeting. Our patented technology offers a breakthrough solution through self-assembling ferritin nanoparticles derived from insect proteins. These nanoparticles are designed to co-assemble with trimeric antigens, providing an innovative platform for enhanced antigen presentation and improved immune responses in vaccines and immunotherapies.

Current Limitations in Antigen Delivery and Display Systems

One of the main hurdles in developing effective vaccines and immunotherapies is finding a reliable delivery method that ensures the proper presentation of antigens to the immune system. Conventional approaches sometimes struggle with antigen stability, appropriate folding, and achieving high immunogenicity. These limitations can reduce the effectiveness of the vaccine or therapeutic treatment, particularly when dealing with complex antigens that require specific configurations to elicit a strong immune response.

In the pharmaceutical and biotechnology industries, there is a growing demand for advanced delivery platforms that can overcome these challenges, providing stable, consistent, and immunogenic antigen display systems. Nanoparticles have emerged as a promising solution for improving antigen delivery, but the development of self-assembling systems that can co-display multiple antigens remains a cutting-edge area of research.

Self-Assembling Nanoparticles for Effective Antigen Display

Our patented system introduces self-assembling insect ferritin nanoparticles as an innovative platform for displaying co-assembled trimeric antigens. Ferritin, a naturally occurring protein complex, has the unique ability to form nanoparticles that are stable, biocompatible, and highly structured. By leveraging ferritin derived from insect sources, this technology enables the assembly of nanoparticles that efficiently present trimeric antigens, ensuring that the antigens are correctly folded and displayed in a highly immunogenic configuration.

The self-assembling nature of the system ensures that the nanoparticles are produced consistently and reliably, providing a scalable solution for vaccine production. This technology has broad applications in the development of next-generation vaccines for infectious diseases, cancer immunotherapies, and other therapeutic areas that require precise antigen delivery and robust immune responses. Additionally, the use of trimeric antigens allows for the targeting of more complex pathogens and diseases, improving the efficacy of the treatment.

Key Benefits

Enhanced Antigen Presentation: Provides a stable and structured platform for displaying trimeric antigens, improving immune responses.
Self-Assembling Nanoparticles: Offers a reliable and scalable system for consistent nanoparticle production.
Broad Therapeutic Applications: Suitable for vaccine development, cancer immunotherapy, and other immunological treatments.
Improved Stability and Immunogenicity: Ensures that antigens are correctly folded and presented in an immunogenic configuration.

Empowering the Next Generation of Vaccines and Immunotherapies

Licensing this self-assembling nanoparticle technology provides pharmaceutical and biotechnology companies with a powerful tool for advancing vaccine development and immunotherapy. With its unique ability to present antigens in a structured and stable format, this system opens new possibilities for creating highly effective, next-generation treatments for a wide range of diseases.

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Abstract
Claims

Disclosed are recombinant insect ferritin nanoparticles that can be used to display two different trimeric antigens at an equal ratio. Also disclosed are nucleic acids encoding the recombinant insect ferritin nanoparticles and methods of producing the recombinant insect ferritin nanoparticles. Methods for eliciting an immune response in a subject are also provided.

1. A recombinant insect ferritin nanoparticle, comprising:

twelve recombinant insect ferritin heavy chain fusion proteins and twelve recombinant insect ferritin light chain fusion proteins self-assembled into a globular nanoparticle; and

eight self-assembled heterologous trimeric antigens extending radially outward from the outer surface of the globular nanoparticle, the eight heterologous trimeric antigens comprising four trimers of a first protein and four trimers of a second protein having a different amino acid sequence from the first protein; wherein

the recombinant insect ferritin nanoparticle comprises a shape having a tetrahedral symmetry;

the recombinant insect ferritin heavy chain fusion proteins comprise an N-terminal fusion of the first protein to a recombinant insect ferritin heavy chain; and

the recombinant insect ferritin light chain fusion proteins comprise an N-terminal fusion of the second protein to a recombinant insect ferritin light chain.

2. The recombinant insect ferritin nanoparticle of claim 1, wherein the insect ferritin heavy and light chains are Trichoplusia ni ferritin heavy and light chains.

3. The recombinant insect ferritin nanoparticle of claim wherein:

the recombinant ferritin heavy chains comprise 172 to 174 amino acids from the C-terminus of an insect ferritin heavy chain and a deletion of the remaining N-terminal amino acids; and

the recombinant ferritin light chains comprise 182 to 184 amino acids from the C-terminus of an insect ferritin light chain and a deletion of the remaining N-terminal amino acids.

4. The recombinant insect ferritin nanoparticle of claim 1, wherein:

(A) the recombinant ferritin heavy chains comprise an amino acid sequence at least 90% identical to SEQ ID NO: 2; and the recombinant ferritin light chains comprise an amino acid sequence at least 90% identical to SEQ ID NO: 6; or

(B) the recombinant ferritin heavy chains comprise an amino acid sequence at least 90% identical to SEQ ID NO: 4; and the recombinant ferritin light chains comprise an amino acid sequence at least 90% identical to SEQ ID NO: 8.

5. The recombinant insect ferritin nanoparticle of claim 1, wherein:

(A) the recombinant ferritin heavy chains comprise or consist of the amino acid sequence set forth as SEQ ID NO: 2, and the recombinant ferritin light chains comprise or consist of the amino acid sequence set forth as SEQ ID NO: 6; or

(B) the recombinant ferritin heavy chains comprise or consist of the amino acid sequence set forth as SEQ ID NO: 4, and the recombinant ferritin light chains comprise or consist of the amino acid sequence set forth as SEQ ID NO: 8.

6. The recombinant insect ferritin nanoparticle of claim 1, wherein the trimeric antigens are viral envelope protein ectodomain trimers and the first protein and the second protein comprise a first viral envelope protein ectodomain and a second viral envelope protein, respectively.

7. The recombinant insect ferritin nanoparticle of claim 6, wherein the first and second viral envelope protein ectodomains are fused to the recombinant ferritin heavy and light chains by a peptide linker.

8. The recombinant insect ferritin nanoparticle of claim 6, wherein the first and second viral envelope protein ectodomains are from two different strains of the same virus.

9. The recombinant insect ferritin nanoparticle of claim 8, wherein the virus is any one of HIV-1, influenza, RSV, MPV, HPIV, Ebola, Marburg, MERS coronavirus, or SARS coronavirus.

10. The recombinant insect ferritin nanoparticle of claim 6, wherein the first and second viral envelope protein ectodomains comprise:

HIV-1 Env ectodomains from two different strains of HIV-1;

influenza HA ectodomains or HA stems from two different strains of influenza;

influenza HA stems from two different strains of influenza;

RSV F ectodomains from two different strains of RSV;

MPV F ectodomains from two different strains of MPV;

Ebola GP ectodomains from two different strains of Ebola virus; or

Coronavirus S protein ectodomains from two different strains of MERS or SARS coronavirus.

11. The recombinant insect ferritin nanoparticle of claim 10, wherein the first and second viral envelope protein ectodomains comprise:

HIV-1 Env ectodomains from two different strains of HIV-1, and wherein the HIV-1 Env ectodomains comprise an HIV-1 gp120 polypeptide and an HIV-1 gp41 ectodomain;

influenza HA ectodomains from two different strains of influenza, and wherein the influenza HA ectodomains comprise an HA1 polypeptide and a HA2 ectodomain;

RSV F ectodomains from two different strains of RSV, and wherein the RSV F ectodomains comprise an RSV F₂polypeptide and a RSV F1 ectodomain; or

MPV F ectodomains from two different strains of MPV, and wherein the MPV F ectodomains comprise an MPV F₂polypeptide MPV F1 ectodomain.

12. The recombinant insect ferritin nanoparticle of claim 10, wherein the first and second viral envelope protein ectodomains comprise HIV-1 Env ectodomains from two different strains of HIV-1, and wherein the HIV-1 Env ectodomains are stabilized in a prefusion mature closed conformation by a non-natural disulfide bond between cysteine substitutions at positions 201 and 433, a non-natural disulfide bond between cysteine substitutions at positions 501 and 605, and a proline substitution at position 559.

13. The recombinant insect ferritin nanoparticle of claim 10, wherein the first and second viral envelope protein ectodomains comprise HIV-1 Env ectodomains from two different strains of HIV-1, and wherein:

the recombinant insect ferritin heavy chain fusion protein comprises the HIV-1 Env ectodomain and insect ferritin heavy chain set forth in one of SEQ ID NOs: 28-52, 55-63, 71, or 73-78; and/or

the recombinant insect ferritin light chain fusion protein comprises the HIV-1 Env ectodomain and insect ferritin light chain set forth in one of SEQ ID NOs: 26-27, 53-54, 64-70, 72, or 79-84.

14. The recombinant insect ferritin nanoparticle of claim 10, wherein the first and second viral envelope protein ectodomains comprise influenza HA ectodomains or recombinant influenza HA stems from two different strains of influenza, and wherein:

the recombinant insect ferritin heavy chain fusion protein comprises the insect ferritin heavy chain and influenza HA ectodomain or recombinant influenza HA stem and set forth in one of SEQ ID NOs: 129-134 or 166, 169, 172, 175, 177, 180, or 182;

the recombinant insect ferritin light chain fusion protein comprises the insect ferritin light chain and influenza HA ectodomain or recombinant influenza HA stem and set forth in one of SEQ ID NOs: 135-140, 167-168, 170-171, 173-174, 176, 179-179, 181, or 182; and/or

the recombinant insect ferritin heavy chain fusion protein comprises the insect ferritin heavy chain and recombinant influenza HA stem and set forth in one of SEQ ID NOs: 166, 169, 172, 175, 177, 180, or 182; and/or the recombinant insect ferritin light chain fusion protein comprises the insect ferritin light chain and recombinant influenza HA stem and set forth in one of SEQ ID NOs: 167-168, 170-171, 173-174, 176, 179-179, 181, or 182.

15. The recombinant insect ferritin nanoparticle of claim 10, wherein the first and second viral envelope protein ectodomains comprise RSV F ectodomains from two different strains of RSV, and wherein the RSV F ectodomains are stabilized in a prefusion conformation by a non-natural disulfide bond between cysteine substitutions at positions 155 and 290, and S190F and V207L cavity filling substitutions.

16. The recombinant insect ferritin nanoparticle of claim 10, wherein the first and second viral envelope protein ectodomains comprise RSV F ectodomains from two different strains of RSV, and wherein:

the recombinant insect ferritin heavy chain fusion protein comprises the RSV F ectodomain and insect ferritin heavy chain as set forth in one of SEQ ID NOs: 151-152, or 155-156; and/or

the recombinant insect ferritin light chain fusion protein comprises the RSV F ectodomain and insect ferritin light chain as set forth in one of SEQ ID NOs: 149-150 or 153-154.

17. The recombinant insect ferritin nanoparticle of claim 10, wherein the first and second viral envelope protein ectodomains comprise MPV F ectodomains from two different strains of MPV, and wherein the MPV F ectodomains are stabilized in a prefusion conformation by a non-natural disulfide bond between cysteine substitutions at positions 113 and 339, and T160F and I177L cavity filling substitutions.

18. The recombinant insect ferritin nanoparticle of claim 1, wherein the ectodomain is a single chain ectodomain.

19. The recombinant insect ferritin nanoparticle of claim 1, wherein administration of an effective amount of the recombinant ferritin nanoparticle to a subject induces an immune response to the trimeric antigens in the subject.

20. A recombinant insect ferritin nanoparticle, comprising:

twelve recombinant ferritin heavy chains and twelve recombinant ferritin light chains self-assembled into a globular ferritin nanoparticle; wherein

the recombinant ferritin heavy chains comprise an amino acid sequence at least 90% identical to SEQ ID NO: 2; and the recombinant ferritin light chains comprise an amino acid sequence at least 90% identical to SEQ ID NO: 6; or

the recombinant ferritin heavy chains each comprise an amino acid sequence at least 90% identical to SEQ ID NO: 3; and the recombinant ferritin light chains each comprise an amino acid sequence at least 90% identical to SEQ ID NO: 8.

21. The recombinant insect ferritin nanoparticle of claim 20, wherein

the recombinant ferritin heavy chains comprise or consist of the amino acid sequence set forth as SEQ ID NO: 2, and the recombinant ferritin light chains comprise or consist of the amino acid sequence set forth as SEQ ID NO: 6; or

the recombinant ferritin heavy chains comprise or consist of the amino acid sequence set forth as SEQ ID NO: 3, and the recombinant ferritin light chains comprise or consist of the amino acid sequence set forth as SEQ ID NO: 8.

22. An isolated nucleic acid molecule encoding:

the recombinant insect ferritin light chain fusion protein, the recombinant insect ferritin heavy chain fusion protein, the recombinant insect ferritin light chain, and/or the recombinant insect ferritin heavy chain of claim 1.

23. An isolated nucleic acid molecule encoding:

a recombinant insect ferritin heavy chains comprising 172 to 174 amino acids from the C-terminus of an insect ferritin heavy chain and a deletion of the remaining N-terminal amino acids; and/or

a recombinant insect ferritin light chains comprising 182 to 184 amino acids from the C-terminus of an insect ferritin light chain and a deletion of the remaining N-terminal amino acids.

24. The nucleic acid molecule of claim 22, operably linked to a promoter.

25. A expression vector comprising the nucleic acid molecule of claim 24.

26. An isolated host cell comprising the expression vector of claim 25.

27. A method of producing a recombinant insect ferritin nanoparticle, comprising:

transfecting a permissive cell culture with the vector of claim 25;

incubating the cell culture for a sufficient period of time to allow for protein expression from the vector; and

purifying the insect ferritin nanoparticle.

28. An immunogenic composition comprising an effective amount of the recombinant insect ferritin nanoparticle of claim 1, and a pharmaceutically acceptable carrier.

29. A method of eliciting an immune response to a trimeric antigen in a subject, comprising administering to the subject an effective amount of the recombinant insect ferritin nanoparticle of claim 1 to generate the immune response.

30. A method for treating or inhibiting a viral infection in a subject, comprising administering to the subject a therapeutically effective amount of the recombinant insect ferritin nanoparticle of claim 6 to induce an immune response that treats or inhibits the viral infection in the subject.

31. The method of claim 29, comprising administering 10-300 μg of the recombinant insect ferritin nanoparticle to the subject to induce the immune response.

32. The method of claim 29, wherein the recombinant insect ferritin nanoparticle comprises trimeric antigens comprising:

HIV-1 Env ectodomains from two different strains of HIV-1, wherein the immune response inhibits or treats HIV-1 infection;

influenza HA ectodomains or recombinant influenza HA stems from two different strains of influenza, wherein the immune response inhibits or treats influenza infection;

RSV F ectodomains from two different strains of RSV, wherein the immune response inhibits or treats RSV infection; or

MPV F ectodomains from two different strains of MPV, wherein the immune response inhibits or treats MPV infection.

33. The method of claim 29, comprising a prime-boost administration of the immunogenic composition.

34.-35. (canceled)

Title

Self-assembling insect ferritin nanoparticles for display of co-assembled trimeric antigens

Inventor(s)

Peter Kwong, Ivelin Georgiev, Michael Gordon Joyce, Masaru Kanekiyo, Aliaksandr Druz, Ulrich Baxa, Joseph Van Galen, Rita Chen, Cheng Cheng, John Mascola, Yaroslav Tsybovsky, YongPing Yang, Paul Thomas, Barney Graham, Syed Mohammad Moin, Jeffrey Boyington, Kizzmekia Corbett

Assignee(s)

US Department of Health and Human Services

Patent #

20190330279

Patent Date

October 31, 2019